The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side-chain dynamics of the a-helical sensory rhodopsin II and the b-barrel outer membrane protein Wh ave been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques.Despite their differing topologies,b oth proteins have as imilar distribution of methyl-bearing side-chain motion that is largely independent of membrane mimetic.T he methyl-bearing side chains of both proteins are,o na verage,m ore dynamic in the ps-ns timescale than any soluble protein characterized to date. Accordingly,b oth proteins retain an extraordinary residual conformational entropyi nt he folded state,w hichp rovides ac ounterbalance to the absence of the hydrophobic effect. Furthermore,t he high conformational entropyc ould greatly influence the thermodynamics underlying membrane-protein functions,i ncluding ligand binding,a llostery,and signaling.
Solution NMR continues to make strides in addressing protein systems of significant size and complexity. A fundamental requirement to fully exploit the N-H TROSY and C-H methyl TROSY effects is highly deuterated protein. Unfortunately, traditional overexpression in Escherichia coli (E. coli) during growth on media prepared in DO leads to many difficulties and limitations, such as cell toxicity, decreased yield, and the need to unfold or destabilize proteins for back exchange of amide protons. These issues are exacerbated for non-ideal systems such as membrane proteins. Expression of protein during growth in HO, with the addition of H-labeled amino acids derived from algal extract, can potentially avoid these issues. We demonstrate a novel fermentation methodology for high-density bacterial growth in HO M9 medium that allows for appropriate isotopic labeling and deuteration. Yields are significantly higher than those achieved in DO M9 for a variety of protein targets while still achieving 75-80% deuteration. Because the procedure does not require bulk DO or deuterated glucose, the cost per liter of growth medium is significantly decreased; taking into account improvements in yield, these savings can be quite dramatic. Triple-labeled protein is also efficiently produced including specific CH labeling of isoleucine, leucine, and valine using the traditional ILV precursors in combination with an ILV-depleted mix of H/N amino acids. These results are demonstrated for the membrane protein sensory rhodopsin II and the soluble proteins human aldoketoreductase AKR1c3, human ubiquitin, and bacterial flavodoxin. Limitations of the approach in the context of very large molecular weight proteins are illustrated using the bacterial Lac repressor transcription factor.
The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side‐chain dynamics of the α‐helical sensory rhodopsin II and the β‐barrel outer membrane protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Despite their differing topologies, both proteins have a similar distribution of methyl‐bearing side‐chain motion that is largely independent of membrane mimetic. The methyl‐bearing side chains of both proteins are, on average, more dynamic in the ps–ns timescale than any soluble protein characterized to date. Accordingly, both proteins retain an extraordinary residual conformational entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect. Furthermore, the high conformational entropy could greatly influence the thermodynamics underlying membrane‐protein functions, including ligand binding, allostery, and signaling.
There are several large-scale efforts to compare the sensitivities of tumor cells to chemotherapeutic drugs and small-molecule pathway probes. However, to date, none of these profiling efforts systematically probed malignant peripheral nerve sheet tumors (MPNST). We initiated a database of drug sensitivities of MPNST as part of a course in high-throughput screening. We first developed a panel of 130 drugs highly relevant to neurofibromatosis (NF1 and NF2) that included a comprehensive set of MEK, RAF, RAS, FTI, PAK, ERK inhibitors, a representative set of drugs against many other cancer pathways including Wnt, Hedgehog, p53, EGF, HDAC, as well as classical cytotoxic agents such as doxorubicin and taxol. Many of the drugs in our panel are in clinical trials themselves, or closely related to drugs in trials for NF1. The drugs were tested against cells in 384-well plates at eight concentrations ranging from .004 μM to 10 μM. Cells were allowed to attach overnight, incubated for 72 hours with drugs, and then analyzed for ATP content using ATPlite. To date, we have profiled nine MPNST cell lines (ST88-14, ST88-3, 90-8, SNF02.2, STS26, T265, S462TY, SNF96.2, SNF94.3) and one NF2 Schwannoma cell line (HEI193). We also tested several cell lines that were tested in other screens so that we can compare our results to the other databases. The IC50 was calculated as a common measure of how effective each drug is using GraphPad Prism software. NF1 cells were distinguished from NF2 cells and STS26 cells (a spontaneous MPNST cell line derived from a patient who did not have NF1) by their strong sensitivity to MEK and Bromodomain inhibitors. Some drugs, including cytotoxic agents, Pak inhibitors, and HDAC inhibitors, were broadly toxic, inhibiting growth regardless of NF1 and NF2 status. None of the drugs in our panel exclusively inhibited HEI193 cells. With this study we initiated a database to archive the drug sensitivities of NF1 and NF2 cells that will be expanded in future versions of our course. Sponsored by Children's Tumor Foundation. Citation Format: Jianman Guo, Michael Grovola, Grace Coggins, Patrick Duggan, Jiale Huang, Claire Song, Gabriela Witek, Danny Lin, Hong Xie, David Schultz, Simon Berritt, Jeffrey Field. The NF1 and NF2 pharmacome project, a course in high-throughput screening to identify targets and profile the sensitivity of MPNST cells to candidate drugs [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A29.
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